The invention relates to a process for the inactivation and/or elimination of coated and/or noncoated viruses from a plasma protein solution.
As is known, human plasma is a raw material for a number of modern therapeutics. These include albumin, the immunoglobulins and the group of blood clotting factors.
The need for blood clotting factors results from the necessity that patients with congenital deficiencies of individual clotting factors can only survive by substitution of these factors. In addition, however, there are also acquired deficiencies of blood clotting factors, which likewise make substitution necessary.
Although it was initially assumed that the substitution of plasma proteins is without risk for the patient, it soon emerged that as a result of therapy with preparations of this type infectious diseases can be transmitted from the plasma donor, which in many cases are caused by contamination with disease-inducing viruses. The object was therefore to make available, by inactivation and/or elimination of viruses of this type, highly purified plasma preparations and preparations of blood clotting factors which can be used in patients without danger of a virus infection. A number of processes have already been proposed for this.
Thus, European Patent 0 124 506 discloses a process for the inactivation of proliferative pathogens in preparations which contain plasma enzymes, clotting factors, immunoglobulins or other proteins present in the blood, in which the preparation is brought to a salt concentration of more than 0.5 molar by addition of ammonium sulfate and heat-treated, whereupon the salt is removed from the preparation. In this process, ammonium sulfate is added to a preparation having a protein content of up to 30% until a 2- to 4-molar concentration is reached. A heat treatment is then carried out during a period of up to 100 hours and at a temperature of 40 to 121xc2x0 C. Of course, in a process of this type attention must be paid to the fact that in the salt and heat treatment used not only the viruses are inactivated and/or eliminated, but that in this case also the biological activity of the plasma proteins contained in the preparation is retained to as high an extent as possible. A need therefore furthermore exists for processes for the inactivation and/or elimination of viruses from a plasma protein solution, which not only fully exclude the transmission of virus infections by plasma preparations of this type, but moreover are so gentle that the biological activities of the plasma proteins, in particular also of the blood clotting factors, are not adversely affected.
It has now been found that these requirements are fulfilled in an excellent manner by a process for the inactivation and/or elimination of coated and/or noncoated viruses from a plasma protein solution if a plasma protein solution is subjected to incubation at room temperature and alkaline pHs at an ammonium salt concentration of 13 to 22% by weight (=25 to 39% ammonium sulfate saturation), pasteurized at approximately 60xc2x0 C for several hours after separation of the precipitates and the removal of the ammonium salt down to a residual content of less than 0.5 mol/l and then processed to give a therapeutically employable plasma protein preparation.
Plasma proteins within the meaning of the invention are not only the intact, active proteins themselves, but also their precursors. The plasma proteins can be obtained from plasma or other body fluids.
For this process, a plasma protein solution previously purified by affinity chromatography is preferably employed and the process is carried out at a pH of between 8 and 11. The virus reduction takes place either by elimination, e.g. in the case of the canine parvovirus (CPV), where a temperature of 25xc2x11xc2x0 C. is very particularly preferred, or by a combination of elimination and inactivation, e.g. in the case of the bovine virus diarrhea virus (BVDV) or in the case of HIV.
A further improvement in the process according to the invention can be achieved by adding ammonium salt again after the incubation with an ammonium salt concentration of 13 to 22% by weight (=25 to 39% ammonium sulfate saturation) and removal and precipitation of the impurities by centrifugation or filtration, and increasing the concentration to 24 to 27% by weight (=44.4 to 50% ammonium sulfate saturation). In this second precipitation, the plasma protein is precipitated in pure form and then pasteurized at approximately 60xc2x0 C. for several hours after removal of the ammonium salt down to a residual content of less than 0.5 mol/l. The precipitate can then be processed to give a therapeutically employable plasma protein preparation.
2 to 4 hours should be used both for the first and the second incubation. The ammonium salt employed is preferably ammonium sulfate.
In order to obtain a residual activity of the plasma proteins which is as high as possible, it is recommended to carry out the pasteurization in the presence of stabilizers. A suitable stabilizer has proved to be a mixture of sucrose (1000 g/l) with glycine (150 g/l). A high residual activity of the plasma proteins can also be obtained in the presence of sucrose and potassium acetate as stabilizers. In general, the process according to the invention is carried out with plasma proteins obtained from blood and affords a residual activity of up to 95% (based on antithrombin III). However, it can be employed as well also for biotechnologically prepared, recombinant or transgenic plasma proteins which are obtained from single-celled organisms or especially also from transgenic animals.
The maintenance of alkaline pHs is also of particular importance for the process according to the invention. If the pH is increased under otherwise identical conditions, an increase in the virus inactivation can be observed with increasing pH in the protein purification which is achieved by the precipitation reaction. The increased virus reduction can be found both for coated and for noncoated viruses and is attributed to the combination of the effects of ammonium salt and alkaline pH.
It is thus characteristic of the process according to the invention that it contains two virus-reducing process steps, namely pasteurization in aqueous solution (10 hours, 60xc2x0 C.) and ammonium sulfate precipitation. Both virus inactivation steps have high reduction factors in virus reduction. The effect of both steps even on noncoated viruses is of particular importance.